Method of detecting and treating cancer

ABSTRACT

The present invention relates, in part, to newly developed assay for diagnosing cancers, particularly endometrial and mammary, endometriosis and endometrial fibroids along with methods for identifying therapeutic agents that modulate endometrial steroid binding protein II activity for the treatment of the above disorders.

This application is a 371 of PCT/US98/12053, filed on Jun. 9, 1998,which claims priority of appln 60/049,015 filed Jun. 9, 1999.

FIELD OF THE INVENTION

This invention relates, in part, to newly developed assay for diagnosingcancers, particularly endometrial and mammary, endometriosis andendometrial fibroids along with methods for identifying therapeuticagents that modulate Endometrial steroid binding protein II activity fortreatment of the above disorders.

BACKGROUND OF THE INVENTION

Endometrial cancer occurs at a rate of approximately 44,500 new casesper year with approximately 10,000 deaths per year. If diagnosed andtreated early, when the cancer is still confined to the endometrium,cure can be achieved in approximately 95% of the cases by hysterectomy.Pap smears can show endometrial cancers but are effective in only 50% ofthe cases. For the remainder, abnormal vaginal bleeding is typically thefirst clinical sign of endometrial cancer. There is a great need forsensitive methods for the detection of organ-confined endometrialcancer.

Steroid binding proteins, including uteroglobin and CC10, are a class ofproteins which bind steroids along with methylsulfonyl metabolites ofpolychlorinated biphenyls. The exact function of members of this classof protein is uncertain, but uteroglobin has been shown to inhibit PLA₂mediated responses. The gene and gene product of the present inventiondisplay homology to uteroglobin and CC10, show elevated expression ofmRNA in endometrial cancer samples and is expressed in mammary tissue.This gene encoded product will be referred to as Endometrial SteroidBinding Protein II (ESBPII), and their polypeptide and polynucleotidesequences are given in Table 1 and 2, respectively.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide a new method for diagnosing, treating, and monitoringprogression, remission or recurrence of various forms of abnormal cellgrowth, such as cancers, particulary endometrial and mammary cancer, andendometriosis and endometrial fibroids. Further provided are methods toscreen for therapeutic agents and pharmaceutical compositions fortreating abnormal cell growth, such as cancers, particular endometrialand mammary cancer, and endometriosis and endometrial fibroids. Furtherprovided is the utilization of such agents or compositions for thetreatement abnormal cell growth, particulary endometrial and mammarycancer and endometriosis and endometrial fibroids.

Thus, in accordance with one aspect of the present invention there areprovided methods of screening for compounds which bind to and inhibitactivation of the ESBPII.

In accordance with another aspect of the present invention there isprovided a method of using such inhibiting compounds for treatingconditions associated with over-expression of the ESBPII.

In accordance with yet another aspect of the present invention, thereare provided ESBPII antagonists (inhibitors). Among the preferredantagonists are those which mimic ESBPII so as to bind to ESBPII bindingmolecules but not elicit a ESBPII -induced response or more than oneESBPII -induced response. Also among the preferred antagonists aremolecules that bind to or interact with ESBPII so as to inhibit aneffect of ESBPII or more than one effect of ESBPII or which preventexpression of ESBPII.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The present invention relates to diagnostic assays, both quantitativeand qualitative for detecting levels of ESBPII protein or ESBPII mRNA incells, tissues and bodily fluids, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of ESBPII proteincompared to normal control bodily fluids or tissue samples may be usedto detect the presence of cancers, including endometrial and mammarycancer. Assay techniques that can be used to determine levels of geneexpression, such as ESBPII of the present invention, in a sample derivedfrom a host are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, reverse transcriptase PCR (RT-PCR)assays, gridding, immunohistochemistry assays, in situ hybridizationassays, competitive-binding assays, Western Blot analyses and ELISAassays. Among these, ELISAs are frequently preferred to detect a gene'sexpressed protein in biological fluids. An ELISA assay initiallycomprises preparing an antibody, if not readily available from acommercial source, specific to ESBPII, preferably a monoclonal antibody.In addition a reporter antibody generally is prepared which bindsspecifically to ESBPII. The reporter antibody is attached to adetectable reagent such as radioactive, fluorescent or enzymaticreagent, for example horseradish peroxidase enzyme or alkalinephosphatase.

To carry out the ELISA, antibody specific to ESBPII is incubated on asolid support, e.g. a polystyrene dish, that binds the antibody. Anyfree protein binding sites on the dish are then covered by incubatingwith a non-specific protein such as bovine serum albumin. Next, thesample to be analyzed is incubated in the dish, during which time ESBPIIbinds to the specific antibody attached to the polystyrene dish. Unboundsample is washed out with buffer. A reporter antibody specificallydirected to ESBPII and linked to horseradish peroxidase is placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to ESBPII. Unattached reporter antibody is then washedout. Reagents for peroxidase activity, including a colorimetricsubstrate are then added to the dish. Immobilized peroxidase, linked toESBPII antibodies, produces a colored reaction product. The amount ofcolor developed in a given time period is proportional to the amount ofESBPII protein present in the sample. Quantitative results typically areobtained by reference to a standard curve. Without limiting the instantinvention, typically, for a quantitative diagnostic assay a positiveresult indicating the disease is one in which blood levels are higherthan three standard deviations above the mean blood level for a normalhealthy population of individuals (99.86% of the population).

A competition assay may be employed wherein antibodies specific toESBPII attached to a solid support and labeled ESBPII and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of ESBPII in the sample.

Nucleic acid methods may be used to detect ESBPII mRNA as a marker forabnormal cell growth including endometrial cancer, mammary cancer,endometriosis and endometrial fibroids. Polymerase chain reaction (PCR)and other nucleic acid methods, such as ligase chain reaction (LCR) andnucleic acid sequence based amplification (NASABA), can be used todetect malignant cells for diagnosis and monitoring of variousmalignancies. For example, reverse-transcriptase PCR (RT-PCR) is apowerful technique which can be used to detect the presence of aspecific mRNA population in a complex mixture of thousands of other mRNAspecies. In RT-PCR, an mRNA species is first reverse transcribed tocomplementary DNA (cDNA) with use of the enzyme reverse transcriptase;the cDNA is then amplified as in a standard PCR reaction. RT-PCR canthus reveal by amplification the presence of a single species of mRNA.Accordingly, if the mRNA is highly specific for the cell that producesit, RT-PCR can be used to identify the presence of a specific type ofcell.

Hybridization to clones arrayed on a grid can be used to both detect theexpression of and quantitate the level of expression of that gene(gridding). In this approach, a cDNA encoding the ESBP II gene is fixedto a substrate. The substrate may be of any suitable type including butnot limited to glass, nitrocellulose, nylon or plastic. DNA encoding theESBP II clone is attached to the substrate and then incubated with theanalyte, which may be RNA or a complementray DNA (cDNA) copy of the RNA,isolated from the tissue of interest. Hybridization between thesubstrate bound clone and the analyte can be detected and quantitated byseveral means including but not limited to radioactive labeling orfluorescence labeling of the analyte or a secondary molecule designed todetect the hybrid. Quantitation of the level of gene expression can bedone by comparison of the intensity of the signal from the analytecompared with that determined from known standards. The standards can beobtained by in vitro transcription of the target gene, quantitating theyield, and then using that material to generate a standard curve.

The above tests can be carried out on samples derived from patients'bodily fluids and tissue extracts (homogenates or solubilized tissue)such as from blood, urine, saliva, tissue biopsy and autopsy material.

Antibodies

The ESBPII polypetide, its fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of a Fab expression library. Various procedures known in the artmay be used for the production of such antibodies and fragments.

Antibodies generated against ESBPII can be obtained by direct injectionof the polypeptide into an animal or by administering the polypeptide toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptide itself. In this manner, even a sequence encodingonly a fragment of the polypeptide can be used to generate antibodiesbinding the whole native polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature 256:495-497 (1975)), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72 (1983)) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985)).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic ESBPII.

Thus, among others, antibodies against ESBPII may be employed totreat/inhibit various forms of abnormal cell growth, includingendometrial and mammary cancer, along with endometriosis and endometrialfibroids.

ESBPII Binding Molecules and Assays

ESBPII could be used to isolate proteins which interact with it and thisinteraction could be a target for interference. Inhibitors ofprotein-protein interactions between ESBPII and other factors could leadto the development of pharmaceutical agents for the modulation of ESBPIIactivity. As used herein, the term “modulate” refer to affecting theESBPII function.

Thus, this invention also provides a method for identification ofbinding molecules to ESBPII. Genes encoding proteins for bindingmolecules to ESBPII can be identified by numerous methods known to thoseof skill in the art, for example, ligand panning and FACS sorting. Suchmethods are described in many laboratory manuals such as, for instance,Coligan et al., Current Protocols in Immunology I (Rivett, A. J.Biochem. J. 291:1-10 (1993)): Chapter 5 (1991).

For example, the yeast two-hybrid system provides methods for detectingthe interaction between a first test protein and a second test protein,in vivo, using reconstitution of the activity of a transcriptionalactivator. The method is disclosed in U.S. Pat. No. 5,283,173; reagentsare available from Clontech and Stratagene. Briefly, ESBPII cDNA isfused to a Gal4 transcription factor DNA binding domain and expressed inyeast cells. cDNA library members obtained from cells of interest arefused to a transactivation domain of Gal4. cDNA clones which expressproteins which can interact with ESBPII will lead to reconstitution ofGal4 activity and transactivation of expression of a reporter gene suchas Gall-lacZ.

An alternative method is screening of λgt11, λZAP (Stratagene) orequivalent cDNA expression libraries with recombinant ESBPII.Recombinant ESBPII protein or fragments thereof are fused to smallpeptide tags such as FLAG, HSV or GST. The peptide tags can possessconvenient phosphorylation sites for a kinase such as heart musclecreatine kinase or they can be biotinylated. Recombinant ESBPII can bephosphorylated with ³²[P] or used unlabeled and detected withstreptavidin or antibodies against the tags. λgt11cDNA expressionlibraries are made from cells of interest and are incubated with therecombinant ESBPII, washed and cDNA clones isolated which interact withESBPII. See, e.g., T. Maniatis et al, supra.

Another method is the screening of a mammalian expression library inwhich the cDNAs are cloned into a vector between a mammalian promoterand polyadenylation site and transiently transfected in COS or 293 cellsfollowed by detection of the binding protein 48 hours later byincubation of fixed and washed cells with a labelled ESBPII, preferablyiodinated, and detection of bound ESBPII by autoradiography. See Sims etal., Science 241:585-589 (1988) and McMahan et al., EMBO J. 10:2821-2832(1991). In this manner, pools of cDNAs containing the cDNA encoding thebinding protein of interest can be selected and the cDNA of interest canbe isolated by further subdivision of each pool followed by cycles oftransient transfection, binding and autoradiography. Alternatively, thecDNA of interest can be isolated by transfecting the entire cDNA libraryinto mammalian cells and panning the cells on a dish containing ESBPIIbound to the plate. Cells which attach after washing are lysed and theplasmid DNA isolated, amplified in bacteria, and the cycle oftransfection and panning repeated until a single cDNA clone is obtained.See Seed et al, Proc. Natl. Acad. Sci. USA 84:3365 (1987) and Aruffo etal., EMBO J. 6:3313 (1987). If the binding protein is secreted, its cDNAcan be obtained by a similar pooling strategy once a binding orneutralizing assay has been established for assaying supernatants fromtransiently transfected cells. General methods for screeningsupernatants are disclosed in Wong et al., Science 228:810-815 (1985).

Another alternative method is isolation of proteins interacting withESBPII directly from cells. Fusion proteins of ESBPII with GST or smallpeptide tags are made and immobilized on beads. Biosynthetically labeledor unlabeled protein extracts from the cells of interest are prepared,incubated with the beads and washed with buffer. Proteins interactingwith ESBPII are eluted specifically from the beads and analyzed bySDS-PAGE. Binding partner primary amino acid sequence data are obtainedby microsequencing. Optionally, the cells can be treated with agentsthat induce a functional response such as tyrosine phosphorylation ofcellular proteins. An example of such an agent would be a growth factoror cytokine such as interleukin-2.

Another alternative method is immunoaffinity purification. RecombinantESBPII is incubated with labeled or unlabeled cell extracts andimmunoprecipitated with anti-ESBPII antibodies. The immunoprecipitate isrecovered with protein A-Sepharose and analyzed by SDS-PAGE. Unlabelledproteins are labeled by biotinylation and detected on SDS gels withstreptavidin. Binding partner proteins are analyzed by microsequencing.Further, standard biochemical purification steps known to those skilledin the art may be used prior to microsequencing.

Yet another alternative method is screening of peptide libraries forbinding partners. Recombinant tagged or labeled ESBPII is used to selectpeptides from a peptide or phosphopeptide library which interact withESBPH. Sequencing of the peptides leads to identification of consensuspeptide sequences which might be found in interacting proteins.

ESBPII binding partners identified by any of these methods or othermethods which would be known to those of ordinary skill in the art aswell as those putative binding partners discussed above can be used inthe assay method of the invention. Assaying for the presence ofESBPII/binding partner complex are accomplished by, for example, theyeast two-hybrid system, ELISA or immunoassays using antibodies specificfor the complex. In the presence of test substances which interrupt orinhibit formation of ESBPII/binding partner interaction, a decreasedamount of complex will be determined relative to a control lacking thetest substance.

Assays for free ESBPII or binding partner are accomplished by, forexample, ELISA or immunoassay using specific antibodies or by incubationof radiolabeled ESBPII with cells or cell membranes followed bycentrifugation or filter separation steps. In the presence of testsubstances which interrupt or inhibit formation of ESBPII/bindingpartner interaction, an increased amount of free ESBPII or free bindingpartner will be determined relative to a control lacking the testsubstance.

Polypeptides of the invention also can be used to assess ESBPII bindingcapacity of ESBPII binding molecules in cells or in cell-freepreparations.

Agonists and Antagonists—Assays and Molecules

The ESBPII may be employed in a process for screening for compoundswhich either inhibit, promote or modulate the activity of ESBPII.

Examples of potential ESBPII antagonists are small molecules such asorganic molecules or peptides, antibodies, or in some cases anoligonucleotide, which binds to ESBPII and prevents activity.

Potential antagonists also include small molecules or proteins which areclosely related to the binding molecules of the ESBPII, e.g. a fragmentof the binding molecules, which have lost biological function, and whenbind to the ESBPII polypeptide inhibit its activity. “Binding molecules”as used herein refer to molecules that specifcally bind to or interactwith ESBPII polypeptide of the present invention. Included in thedefinition of binding molecules are other factors, co-factors, units orsubunits which enhance ESBPII activity or diminish it. Such bindingmolecules are a part of the present invention. Binding molecules alsomay be non-naturally occurring, such as antibodies and antibody-derivedreagents that bind specifically to ESBPII.

A potential antagonist also includes an antisense construct preparedthrough the use of antisense technology. Antisense technology can beused to control gene expression through triple-helix.formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature ESBPII, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix -see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456(1988); and Dervan et al., Science, 251:1360 (1991)), thereby preventingtranscription and the production of ESBPII polypeptide. The antisenseRNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the ESBPII polypeptide(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that -the antisense RNA or DNA may be expressed in vivo toinhibit production of the ESBPII polypeptide. Included in this deliveryis by gene therapy.

Another potential antagonist is a small molecule which binds to theESBPII making it inaccessible to binding molecules (e.g. substrates)such that normal biological activity is prevented. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules and organic compounds.

This invention additionally provides a method of treating an abnormalcondition related to an excess of ESBPII activity, such as endometrialand mammary cancer, endometriosis and endometrial fibroids, whichcomprises administering to a subject the inhibitor compounds(antagonists) as hereinabove described along with a pharmaceuticallyacceptable carrier in an amount effective to inhibit ESBPII activitydirectly or by blocking binding of binding molecules to ESBPIIpolypeptide.

Compositions and Kits

The compounds which inhibit such ESBPII, may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide or compound, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes, among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Vaccine

Another aspect of the invention relates to a method for inducing animmunological response in an animal, particularly in a mammal, whichcomprises inoculating the animal with ESBPII, or a fragment or variantthereof, adequate to produce antibody to protect said animal fromdiseases of abnormal cell growth such as as endometrial and mammarycancer, endometriosis and endometrial fibroids. Yet another aspect ofthe invention relates to a method of inducing immunological response inan animal which comprises, through gene therapy, delivering geneencoding ESBPII, or a fragment or a variant thereof, for expressingESBPII, or a fragment or a variant thereof in vivo in order to induce animmunological response to produce antibody to protect said animal fromdisease.

Further aspect of the invention relates to an immunological compositionwhich, when introduced into an animal, particularly mammalian host,induces an immunological response in that animal to a given ESBPII geneor protein coded therefrom, wherein the composition comprises arecombinant ESBPHI gene or protein coded therefrom comprising DNA whichcodes for and expresses an antigen of said ESBPII gene or protein codedtherefrom.

The ESBPII or a fragment thereof may be fused with co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradermal etc. injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Whilst the invention has been described with reference to ESBPII, it isto be understood that this covers fragments of the naturally occurringprotein and similar proteins (for example, having sequence homologies of50% or greater) with additions, deletions or substitutions which do notsubstantially affect the immunogenic properties of the recombinantprotein.

The present invention also provides a method for the production oftransgenic animals with altered ESBPII levels for the productions ofanimals bearing ESBPII induced diseases. Transgenic, non-human, animalsmay be obtained by transfecting appropriate fertilized eggs or embryosof a host with nucleic acids encoding the ESBPII disclosed herein, seefor example U.S. Pat. Nos. 4,736,866; 5,175,385; 5,175,384 and5,175,386. The resultant transgenic animal may be used as a model forthe study of altered ESBPII levels. Particularly, useful transgenicanimals are those which display a detectable phenotype associated withthe altered expression of the ESBPII polypeptide. Drugs may then bescreened for their ability to reverse or exacerbate the relevantphenotype.

TABLE I^(a)          10                  30                  50.         .         .         .         .         .MKLSVCLLLVTLALCCYQANAEFCPALVSELLDFFFISEPLFKLSLAKFDAPPEAVAAKL        70                  90          .         .         .GVKRCTDQMSLQKRSLIAEVLVKILKKCSV ^(a)Endometrial Steroid Binding ProteinII (SEQ ID NO: 1)

TABLE II^(b)          10                  30                  50TTGTTTGTGAAAGCTGAGCTCACAGCAAAACAAGCCACCATGAAGCTGTCGGTGTGTCTC        70                  90                 110CTGCTGGTCACGCTGGCCCTCTGCTGCTACCAGGCCAATGCCGAGTTCTGCCCAGCTCTT       130                 150                 170GTTTCTGAGCTGTTAGACTTCTTCTTCATTAGTGAACCTCTGTTCAAGTTAAGTCTTGCC       190                 210                 230AAATTTGATGCCCCTCCGGAAGCTGTTGCAGCCAAGTTAGGAGTGAAGAGATGCACGGAT       250                 270                 290CAGATGTCCCTTCAGAAACGAAGCCTCATTGCGGAAGTCCTGGTGAAAATATTGAAGAAA       310                 330                 350TGTAGTGTGTGACATGTAAAAACTTCATCCTGGTTTCCACTGTCTTTCAATGACACCCTG      370                 390                 410ATCTTCACTGCAGAATGTAAAGGTTTCAACGTCTTGCTTTAATAAATCACTTGCTCTCCAAAAAAAAAAAAA ^(b)Endometrial steroid binding protein II nucleotidesequence (SEQ ID NO: 2)

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples are carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as “Sambrook.”

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow is carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8:4057 (1980).

Example 1

To evaulate the level of ESBPII in normal and endometrial tumor tissue,mRNA was extracted from two endometrial tumors and matched normaltissues. Subsequently, first strand cDNA was prepared with reversetranscriptase and the polymerase chain reaction was done using primersspecific to ESBPII. Results were normalized by using the same amount ofstarting cDNA in each sample. When the resulting products were analyzedby gel electrophoresis followed by ethdium bromide detection, ESBPII wasfound to be expressed in the endometrial tumors at least 5 fold greaterin the endometrial tumor tissue relative to the normal adjacent tissue.

Example 2

To determine the tissue distribution of ESBPII expression, mRNA and cDNAwas prepared from a variety of tissues and the polymerase chain reactionwas done using the ESBPII primers. Results are indicated below:

Endometrium + Endometrial Cancer +++++ Mammary ++ Prostate Absent ColonAbsent Colon Cancer Absent Lung Absent Lung Cancer Absent Liver AbsentLiver Cancer Absent Pancreas Absent Heart Absent Skeletal Muscle AbsentPeripheral Blood Cells Absent

What is claimed is:
 1. A diagnostic method for endometrial cancer,mammary cancer, endometriosis or endometrial fibroids comprising:analyzing for the abnormally high level of ESBPII polypeptide in cells,tissues and bodily fluids.
 2. A method of claim 1 in which thediagnostic process involves ELISA.
 3. A method of claim 1 in which thediagnostic process is immunohistochemistry.
 4. A diagnostic method formammary cancer, endometrial cancer, endometriosis or endometrialfibroids comprising: analyzing for the abnormally high or lowtranscription level of ESBPII in cells, tissues and bodily fluids.
 5. Amethod of claim 4 in which the diagnostic process involves Northern blotanalysis.
 6. A method of claim 4 in which the diagnostic processinvolves in situ hybridization.
 7. A method of claim 4 in which thediagnostic process involves RT-PCR.
 8. A method of claim 4 in which thediagnostic process involves gridding.